Dual-Atom Nanozyme Eye Drops Attenuate Inflammation and Break the Vicious Cycle in Dry Eye Disease

Highlights A dual-atom nanozyme (DAN) was successfully prepared based on Fe and Mn bimetallic single-atom embedded in N-doped carbon material and modified with hydrophilic polymer. The DAN possess excellent enzyme catalytic activity and attenuate dramatically inflammation by inhibiting the reactive oxygen species (ROS)/NLRP3 signal axis. The DAN break the vicious cycle in dry eye disease and is a potential strategy for treating dry eye disease. Supplementary Information The online version contains supplementary material available at 10.1007/s40820-024-01322-7.


S1.2 Characterization
Computed tomography (CT) X-ray diffraction (Empyrean XRD) measurements were carried out using an X-ray diffractometer with a Mn Kα radiation source (45 kV, 40 mA).X-ray photoelectron spectroscopy (XPS) spectra were obtained using an X-ray photoelectron spectrometer (AXIS Supra, Shimadzu/Kratos, England) equipped with an Al Kα radiation source.Calibration was performed with adsorption C 1s 284.8 eV as the standard.The appearance of the samples was observed using a field emission scanning electron microscope (FESEM, Auriga FIB, Zeiss, Germany).An FEI TalosF200S transmission electron microscope (TEM; Czech Republic) characterized the morphology and selected area electron diffraction (SAED).HAADF-STEM and high-resolution STEM (HR-STEM) were performed on a JEOL ARM-300F with spherical aberration correction operated at 300 kV.Metal element contents were confirmed by inductively coupled plasma mass spectrometry (ICP-MS; NWR-213).The scanning electron microscope (SEM) images were obtained by a scanning electron microscope (ZEISS Sigma 500, USA).

S1.3 XAFS measurements and simulations
Data reduction, data analysis, and EXAFS fitting were performed and analyzed with the Athena and Artemis programs of the Demeter data analysis packages that utilizes the FEFF6 program to fit the EXAFS data [S1, S2].The energy calibration of the sample was conducted through standard and Fe foil and Mn foil, which as a reference was simultaneously measured.A linear function was subtracted from the pre-edge region, then the edge jump was normalized using Athena software.The χ(k) data were isolated by subtracting a smooth, third-order polynomial approximating the absorption background of an isolated atom.The k3-weighted χ(k) data were Fourier transformed after applying a HanFeng window function (Δk = 1.0).For EXAFS modeling, the global amplitude EXAFS (CN, R, σ 2 and ΔE0) were obtained by nonlinear fitting, with leastsquares refinement, of the EXAFS equation to the Fourier-transformed data in R-space, using Artemis software, EXAFS of the Fe foil and Mn foil are fitted and the obtained amplitude reduction factor S0 2 value (0.710 and 0.816) was set in the EXAFS analysis to determine the coordination numbers (CNs) in the Fe-O, Fe-Fe and Fe-Mn scattering path in sample.

S1.5 In vitro cytocompatibility
The cytotoxicity test of DAN was investigated in HCE-2 and CCL-20.2 cells using a Cell Counting Kit 8 assay.HCE-2 and CCL-20.2 cells were seeded onto 96-well plates at a density of 8 × 10 3 cells per well and 10 × 10 3 cells per well, respectively, and cultured in 5% CO2 at 37 °C for 24 h.Subsequently, the cells were treated with various concentrations of DAN (0, 0.98, 1.95, 3.9, 7.8, 15.6, 31.25 µg/mL) diluted with DMEM/F12 medium respectively for another 24 h.Then the medium was removed and a fresh medium (100 μL) containing 10% CCK-8 solution was added to each well for an additional 4 h at 37 °C.The optical density (OD values) was measured at 450 nm using an Enzyme Labeler (PerkinElmer EnVision, England).Cells cultured with medium alone were used as control.

S1.6
In vitro hypertonic model HCE-2 cells were seeded in 96-well plates at a density of 8 × 10 3 cells per well and incubated overnight at 37 °C.Then the cells were switched to a serum-free DMEM/F12 medium for 6 h before treatment.Next the cells were treated for 24 h in serum-free medium with iso-and hyper-osmolarity (310, 400, 450 and 500 mOsM), which was achieved by adding 0, 50, 70, or 90 mM sodium chloride (NaCl).The osmolarity of the culture media was measured by osmometer (STY-1A, Tianjin Tianda Technology Co., Ltd.).Then the medium was removed and a fresh serum-free medium (100 μL) containing 10% CCK-8 solution was added to each well for an additional 4 h at 37 °C.The optical density (OD values) was measured at 450 nm using an Enzyme Labeler.Similarly, Cells were washed twice with PBS and stained for 30 min at 37° C with 10 µM DCFH-DA in the dark.The ROS (DCF) fluorescence intensity was detected by cytation5 imaging reader (Bioteck, America).

S1.7 In vitro anti-apoptotic properties
HCE-2 cells were seeded in 96-well plates at a density of 8 × 10 3 cells per well and incubated overnight at 37 °C.Then the cells were pretreated with serum-free DMEM/F12 medium containing different concentrations of DAN (1, 2, 4, 8 µg/mL) for 6 h.Subsequently, the cells were cultured in serum-free DMEM-F12 containing 90 mM NaCl, which was used to create a hypertonic environment (500 mOsM) for an additional 24 h.The ROS inhibitor NAC (10 mM) was added 1 h before NaCl supplementation as a positive control.Then the medium was removed and a fresh serum-free medium (100 μL) containing 10% CCK-8 solution was added to each well for an additional 4 h at 37 °C.The optical density (OD values) was measured at 450 nm using an Enzyme Labeler (PerkinElmer EnVision, England).Cells cultured with Nano-Micro Letters S4/S10 medium alone were used as control.

Table S1
EXAFS fitting parameters at the Fe and Mn K-edge for various samples CN, coordination number; b R, the distance to the neighboring atom; c σ 2 , the Mean Square Relative Displacement (MSRD); d ΔE0, inner potential correction; R factor indicates the goodness of the fit. a